Micropropagation is the process of growing new generation plants from a single tissue sample that has been excised from a carefully selected parent plant or cultivar. This process permits the mass reproduction of plants having certain desirable traits since substantially all of the new generation plants produced are genetically identical to and have all the desirable traits of the parent.
Tissue culturing is the process of growing cells in vitro and is used to grow both plant and animal cells. Tissue culturing techniques are commonly used in the early stages of the plant micropropagation process where it is desirable to rapidly produce plant cells. Improvements in tissue culturing techniques also have applications beyond the micropropagation of plants. Essentially the same culturing process is used to culture animal and even human tissue, such tissue being used in the fields of animal agriculture and human and veterinary medicine. Culturing of organic material other than plant and animal cells and tissue, such as bacteria, viruses and algeas, is also performed in vitro for both research and commercial purposes. Improvements in the procedures and apparatus used to reproduce and maintain these organisms would be beneficial, for example, to researchers and industry who require a large or steady supply of such material.
There are problems associated with the prior art culturing apparatus and processes One of the primary problems is contamination. Any of a wide variety of microorganisms, including viruses, bacteria, fungus, molds, yeast and single cell algae, can ruin the cultures during any of the various stages. The smallest of these biological contaminants are the viruses, the largest are the single cell algae. A virus typically ranges in size from 0.1 to 0.45 micrometers although it is suspected that portions of the virus which are as small as 0.01 micrometers may separate from the virus and alone cause contamination. Bacteria typically range in size from 5 to 100 micrometers, while fungi and molds are usually larger than 100 micrometers. Yeast is larger than bacteria, with single cell algae, the largest of these biological contaminants, being larger than yeast.
The prior art sterilized glass or plastic culture containers such as test tubes, flasks or bottles, utilized in conventional culturing technology have serious drawbacks. For example, since plants require both carbon dioxide and oxygen to live and grow, these containers must provide a means for gas exchange. The walls of these traditional glass and plastic containers, however, do not permit the required gaseous interchange. Thus, rubber stoppers having cotton packing or some similar filter material, loosely fitting caps, or baffled plastic caps have been employed to allow an adequate exchange of gas between the tissue or plant and the ambient atmosphere and environment. However, such devices restrict the amount and rate of gas which can be exchanged. Further, such caps and stoppers do not totally protect the plant from contamination by microorganisms such as viruses, bacteria and fungi. Thus, it has been of paramount importance that the tissue culture room and laboratory be kept extremely clean and their atmospheres filtered. Further, precise temperature, humidity, and light conditions must be maintained in the culture room. Gas exchange is also required for culturing animal cells and for certain other microorganisms. Traditional flasks, petrie dishes and the like, while allowing for a certain degree of gas exchange, also allow contamination to occur.
The original cost of the traditional glass or plastic culture containers; the labor and equipment cost to maintain the sterility of the containers; and the added cost of the facilities, equipment, and related conditions required to maintain a sterile growing environment, all represent major cost factors associated with the use of such containers in conventional culturing processes.
The present invention overcomes many of the deficiencies of the prior art techniques of culturing by having the following advantages:
(1) enhanced protection from contamination;
(2) increased growth rates;
(3) no requirement for a sterile culture room;
(4) no requirement for expensive glass containers or the incurrence of replacement costs due to breakage;
(5) no labor cost associated with cleaning and sterilizing containers for reuse;
(6) an increase in the number of plantlets from a culture;
(7) a reduction by approximately one-half the amount of media required in each plant culture;
(8) the elimination of the requirement of strict humidity control in the culture room;
(9) an increase in the number of cultures which can be produced in the same size culture room;
(10) a reduction in the size of the media preparation area and in the size of the autoclave; and
(11) an increase in the number of new cultures which can be established by a laboratory technician.
Other objects and advantages of the invention will appear from the following description.